Type 1 diabetes (T1D) is a complex disease that is mediated by T cell entry into pancreatic islets and the subsequent destruction of insulin-producing b cells. The mechanisms that control these events are poorly understood, a greater understanding of which could lead to the development of novel therapeutic strategies. A significant limitation in addressing these complex issues has been the availability of appropriate genetic tools. We have developed a new approach for the rapid generation of TCR Tg mice, which we refer to as retrogenic (Rg) mice. We will use a panel of TCRs specific for two primary autoantigens of considerable interest, insulin (Ins) and chromogranin A (ChgA), that possess a very broad range of insulitogenic and diabetogenic potentials, novel mouse models and innovative, cutting-edge techniques to determine the biophysical and temporal parameters that regulate CD4+ T cell insulitogenicity and diabetogenicity. This is the primary focus and long-term objective of this project. The simplest hypothesis is that 'highly diabetogenic CD4+ T cells are optimally generated and activated in the pancreatic lymph node (PLN), and have a unique biophysical, kinetic and migratory signature which dictates their pathogenicity'. This hypothesis will be tested and parameters defined in two highly integrated Aims: Aim 1: Biophysical parameters that influence CD4+ T cell diabetogenicity. (A) Do the biophysical properties of the TCR dictate the insulitogenic and diabetogenic potential of CD4+ T cells? In collaboration with Brian Evavold (Emory Univ.), we will determine the 2D affinity, association (on) rate, and dissociation (off) rate of the Ins- and ChgA-specific TCRs for their cognate peptide-MHC complex (pMHC). Together with the sensitivity of TCR Rg T cells in functional assays, we will determine if these biophysical parameters dictate their diabetogenic potential. (B) Does antigen availability affect CD4+ T cell islet entry and diabetogenicity? We will generate NOD mice in which the b cell-restricted expression of a model autoantigen, GAD65, can be externally controlled. We will assess how antigen expression levels and TCR functional avidity cooperate to influence T cell pathogenicity. Aim 2: Temporal parameters that influence CD4+ T cell diabetogenicity. (A) Does the kinetics of CD4+ T cell islet entry determine diabetogenic potential? We will compare the kinetics of T cell islet infiltration, assessed by flow cytometric and histological analysis, and the rate of b cell destruction, determined using non- invasive, bioluminescent imaging of live mice. (B) Do the intra-islet migratory characteristics of CD4+ T cells dictate diabetogenic potential? In collaboration with Alexander Chervonsky (Univ. Chicago), multiphoton intravital microscopy will be used to track CD4+ T cell migration within the islets to determine whether there is a correlation between their migratory characteristics and their insulitogenic and diabetogenic potential. PUBLIC HEALTH RELEVANCE: Type 1 diabetes (T1D) is a complex disease that is mediated by T cell entry into pancreatic islets and the subsequent destruction of insulin-producing b cells. The molecules and mechanisms that control these events are poorly understood. Their identification is of critical importance and could lead to the development of novel therapeutic strategies to cure or limit T1D and improve human health.